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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
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Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation.

Guoquan Wang1, Rui Cheng2, Qiubing Chen1

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A novel VSW-3 RNA polymerase (RNAP) significantly reduces double-stranded RNA (dsRNA) by-products during in vitro transcription (IVT). This leads to lower inflammatory responses and enhanced protein expression for mRNA therapeutics.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • In vitro transcription (IVT) is crucial for mRNA therapeutics.
  • T7 RNA polymerase (RNAP) generates double-stranded RNA (dsRNA) by-products during IVT, activating innate immune responses.
  • dsRNA by-products can limit the efficacy of mRNA therapeutics.

Purpose of the Study:

  • To evaluate a new VSW-3 RNAP for reducing dsRNA production during IVT.
  • To assess the inflammatory potential of mRNA produced using VSW-3 RNAP.
  • To compare protein expression levels of mRNA synthesized by VSW-3 RNAP versus T7 RNAP.

Main Methods:

  • In vitro transcription using VSW-3 RNAP and T7 RNAP.
  • Quantification of dsRNA by-products.
  • Assessment of cellular inflammatory responses to mRNA transcripts.
  • Measurement of protein expression in cell lines (Hela) and animal models (mice).

Main Results:

  • VSW-3 RNAP significantly reduced dsRNA production compared to T7 RNAP.
  • mRNA produced with VSW-3 RNAP showed lower inflammatory stimulation in cells.
  • VSW-3 RNAP transcripts yielded higher protein expression (14-fold in Hela cells, 5-fold in mice) without modified nucleotides.
  • VSW-3 RNAP improved protein production efficacy.

Conclusions:

  • VSW-3 RNAP is a promising alternative to T7 RNAP for mRNA production.
  • This new RNAP minimizes dsRNA by-products, reducing immunogenicity.
  • VSW-3 RNAP enhances protein expression, offering a valuable tool for mRNA therapeutics development.